Project Summary/Abstract The Acid Sensing Ion Channel 1a (ASIC1a) is expressed throughout both central and peripheral nervous systems and has been implicated in a variety of neurological processes. This homotrimeric channel responds to extracellular acidosis with fast activation of an inward cationic current followed by rapid desensitization. Most recently, ASIC1a has emerged as a regulator of synaptic plasticity as well as an important therapeutic target for preventing ischemia-induced central nervous system damage common to both stoke and traumatic brain injury. Importantly, a high-resolution structure of the resting (closed) ASIC1a channel and a detailed understanding of the channel's pH-dependent gating mechanism have remained elusive. The overall goal of this proposal is to address these major gaps in our understanding of the structure, function, and modulation of the ASIC1a channel. Previously solved crystal structures of ASIC1a highlight distinct structural conformations associated with both open and desensitized functional states. These results demonstrated that regions of the trimeric channel, primarily thumb, palm, and wrist domains, exhibit structurally dynamic and state-dependent behavior potentially important for channel gating. Additionally, though ASIC1a is primarily Na+-selective, the channel is slightly permeable to and modulated by extracellular Ca2+. Intriguingly, all three above-mentioned gating domains have been implicated in the Ca2+-dependent modulation of ASIC1a activity. It is currently thought that these Ca2+-dependent modulatory effects may have their roots in disruption of channel gating mechanics. A detailed molecular mechanism for this modulation, however, has yet to be determined. The primary goal of this proposal is to utilize x-ray crystallography experiments to determine the high pH, resting state structure of ASIC1a, the location of Ca2+ binding sites, and the channel's pH-dependent gating mechanism. In support of this goal, a combination of whole-cell patch clamp electrophysiology and isothermal titration calorimetry will be used to further characterize the modulatory interaction between ASIC1a and extracellular Ca2+. The inherent difficultly of membrane protein structural biology makes a complete structural representation covering all functional states of an ion channel very rare. At a basic level the work I am proposing will expand our knowledge of ion channel structure/function relationships and improve our understanding of the highly complex regulatory mechanisms present at central nervous system synapses. Additionally, the information gained from this proposal could provide details important for the development of neuroprotective agents used to treat conditions associated with central ischemia including stoke and traumatic brain injury.